Information on EC 1.5.1.34 - 6,7-dihydropteridine reductase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
1.5.1.34
-
RECOMMENDED NAME
GeneOntology No.
6,7-dihydropteridine reductase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
a 5,6,7,8-tetrahydropteridine + NAD(P)+ = a 6,7-dihydropteridine + NAD(P)H + H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Folate biosynthesis
-
-
L-phenylalanine degradation V
-
-
Metabolic pathways
-
-
phenylalanine metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
5,6,7,8-tetrahydropteridine:NAD(P)+ oxidoreductase
The substrate is the quinonoid form of dihydropteridine. Not identical with EC 1.5.1.3 dihydrofolate reductase.
CAS REGISTRY NUMBER
COMMENTARY hide
70851-99-9
-
9074-11-7
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
monkey
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
sp. ATCC 11299a
-
-
Manually annotated by BRENDA team
strain HB27, gene DHTt
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
-
enzyme catalyzes the last step of tetrahydrobiopterin recycling
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(6R)-L-erythro-dihydrobiopterin + NADH + H+
?
show the reaction diagram
-
-
-
?
(6R)-tetrahydrobiopterin + NAD(P)+
?
show the reaction diagram
-
-
-
r
(6R,S)-6-hydroxy-methyltetrahydropterin + NAD(P)+
?
show the reaction diagram
low activity
-
-
r
(6R,S)-6-methyl-tetrahydropterin + NAD(P)+
?
show the reaction diagram
low activity
-
-
r
(6R,S)-tetrahydrodictyopterin + NAD(P)+
?
show the reaction diagram
-
-
-
r
(6R,S)-tetrahydrofolic acid + NAD(P)+
?
show the reaction diagram
low activity
-
-
r
(6R,S)-tetrahydroneopterin + NAD(P)+
?
show the reaction diagram
low activity
-
-
r
(6S)-tetrahydrobiopterin + NAD(P)+
?
show the reaction diagram
low activity
-
-
r
2-amino-4-hydroxy-6,7-dimethyl-quinonoid-dihydropteridine + NAD(P)H
2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine + NAD(P)+
show the reaction diagram
2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine + NADH
?
show the reaction diagram
-
-
-
-
?
2-amino-4-hydroxy-6-methyl-dihydropteridine + NAD(P)H
2-amino-4-hydroxy-6-methyl-tetrahydropteridine + NAD(P)+
show the reaction diagram
-
-
-
?
2-amino-4-hydroxy-7-methyl-dihydropteridine + NAD(P)H
2-amino-4-hydroxy-7-methyl-tetrahydropteridine + NAD(P)+
show the reaction diagram
-
-
-
?
5,6,7,8-tetrahydropteridine + NAD(P)+
6,7-dihydropteridine + NAD(P)H
show the reaction diagram
enzyme is required for recycling of 6,7-dihydropteridine, in vivo regulation is related to growth phase
-
-
r
6,7-dihydropteridine + NADH
(6R)-L-erythro-5,6,7,8-tetrahydrobiopterin + NAD+
show the reaction diagram
6,7-dimethyl-7,8-dihydropteridine + NADPH
(6R)-L-erythro-6,7-dimethyl-5,6,7,8-tetrahydrobiopterin + NADP+
show the reaction diagram
6,7-dimethyl-7,8-dihydropterin + NADPH + H+
6,7-dimethyl-5,6,7,8-tetrahydropterin + NADP+
show the reaction diagram
6,7-dimethyl-tetrahydropterin + NAD(P)+
?
show the reaction diagram
low activity
-
-
r
6,7-dimethyldihydropterin + NADH + H+
6,7-dimethyltetrahydropterin + NAD+
show the reaction diagram
-
-
-
?
dihydrobiopterin + NADH + H+
tetrahydrobiopterin + NAD+
show the reaction diagram
dihydrofolate + NADPH
tetrahydrofolate + NADP+
show the reaction diagram
-
-
-
-
?
dihydropteroic acid + NAD(P)H
tetrahydropteroate + NAD(P)+
show the reaction diagram
-
-
-
?
dihydropteroylglutamic acid + NAD(P)H
tetrahydropteroylglutamate + NAD(P)+
show the reaction diagram
-
-
-
?
K3Fe(CN)6 + NAD(P)H
K4Fe(CN)6 + NAD(P)+
show the reaction diagram
-
-
-
?
quinoid dihydrobiopterin + NAD(P)H
tetrahydrobiopterin + NAD(P)+
show the reaction diagram
quinonoid (6R)-D-threo-dihydrobiopterin + NADH + H+
?
show the reaction diagram
-
-
-
?
quinonoid 6,6-dimethyldihydropterin + NAD(P)H
6,6-dimethyltetrahydropterin + NAD(P)+
show the reaction diagram
quinonoid 6,7-dimethyl-7,8-(6H)-dihydropterin + NAD(P)H
6,7-dimethyl-5,6,7,8-tetrahydropterin + NAD(P)+
show the reaction diagram
quinonoid 6-methyl-7,8-(6H)-dihydropterin + NAD(P)H
6-methyl-5,6,7,8-tetrahydropterin + NAD(P)+
show the reaction diagram
quinonoid 7,8-(6H)-dihydrobiopterin + NAD(P)H
5,6,7,8-tetrahydrobiopterin + NAD(P)+
show the reaction diagram
quinonoid 7,8-(6H)-dihydrobiopterin + NAD(P)H
5,6,7,8-tetrahydropterin + NAD(P)+
show the reaction diagram
quinonoid dihydro-2-methylamino-4-hydroxy-6,7-dimethylpteridine + NAD(P)H
tetrahydro-2-methylamino-4-hydroxy-6,7-dimethylpteridine + NAD(P)+
show the reaction diagram
-
approx. one-third of activity with 2-amino-4-hydroxy-6,7-dimethyl-quinonoid-dihydropterid
-
?
quinonoid dihydro-L-threo-neopterin + NAD(P)H
tetrahydro-L-threo-neopterin + NAD(P)+
show the reaction diagram
-
-
-
?
quinonoid dihydrobiopterin + NADH + H+
6R-(1'R,2'R)-5,6,7,8-tetrahydrobiopterin + NAD+
show the reaction diagram
quinonoid dihydromonapterin + NAD(P)H
tetrahydromonapterin + NAD(P)+
show the reaction diagram
-
-
-
?
quinonoid dihydroneopterin + NAD(P)H
tetrahydroneopterin + NAD(P)+
show the reaction diagram
-
-
-
?
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
5,6,7,8-tetrahydropteridine + NAD(P)+
6,7-dihydropteridine + NAD(P)H
show the reaction diagram
Q8WTJ2
enzyme is required for recycling of 6,7-dihydropteridine, in vivo regulation is related to growth phase
-
-
r
6,7-dihydropteridine + NADH
(6R)-L-erythro-5,6,7,8-tetrahydrobiopterin + NAD+
show the reaction diagram
P09417
last step of recycling of the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopteridine essential for aromatic amino acid hydroxylases and nitric oxide synthases, regulatory mechanism involve H2O2, enzyme inactivation because of H2O2 accumulation in the epidermis contributes to the pathomechanism of vitiligo
-
-
?
dihydrobiopterin + NADH + H+
tetrahydrobiopterin + NAD+
show the reaction diagram
-
pH 7.4, 37C
-
-
?
quinonoid 7,8-(6H)-dihydrobiopterin + NAD(P)H
5,6,7,8-tetrahydrobiopterin + NAD(P)+
show the reaction diagram
quinonoid dihydrobiopterin + NADH + H+
6R-(1'R,2'R)-5,6,7,8-tetrahydrobiopterin + NAD+
show the reaction diagram
-
-
-
-
?
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
FAD
-
contains bound FAD
FMN
-
one molecule bound per enzyme
NAD(P)H
NADH is the preferred cofactor
additional information
-
no flavin prosthetic group
-
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(R)-2,10,11-trihydroxy-N-n-propylaporphine
(R)-2,10,11-trihydroxyaporphine
(R)-2,11-dihydroxy-10-methoxy-aporphine
(R)-apocodeine
(R)-apomorphine
(R)-N-chloroethylnorapomorphine
(R)-N-hydroxyethylnorapomorphine
(R)-N-n-propylaporphine
(R)-N-n-propylnorapomorphine
(R)-norapomorphine
(S)-2,10,11-trihydroxyaporphine
(S)-bulbocapnine
(S)-N-n-propylnorapomorphine
1-methyl-4-(3',4'-dihydroxyphenyl)-1,2,3,6-tetrahydropyridine
1-methyl-4-(3'-methoxy-4'-hydroxyphenyl)-1,2,3,6-tetrahydropyridine
1-methyl-4-(4'-chlorophenyl)-1,2,3,6-tetrahydropyridine
1-methyl-4-(4'-hydroxyphenyl)-1,2,3,6-tetrahydropyridine
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine
2,4-diamino-7,8-dihydropteroylglutamic acid
-
-
2,4-Diaminopteridine
-
-
2,4-diaminopteroic acid
-
-
2,4-diaminopteroylglutamic acid
-
-
2,6-dichlorophenolindophenol
3',4'-deoxynorlaudanosolinecarboxylic acid
-
noncompetitive vs. quinoid 2-amino-6,7-dimethyl-4-hydroxydihydropteridine and NADH
3(4-hydroxyphenyl)pyruvate
3-iodotyrosine
-
weak inhibition
3-O-methyldopamine
-
0.06 mM, 50% inhibition
3-O-Methylepinephrine
-
0.18 mM, 50% inhibition
3-phenylpyruvic acid
4-(3',4'-dihydroxyphenyl)-1,2,3,6-tetrahydropyridine
4-(3'-methoxy-4'-hydroxyphenyl)-1,2,3,6-tetrahydropyridine
4-(4'-chlorophenyl)-1,2,3,6-tetrahydropyridine
4-(4'-hydroxyphenyl)-1,2,3,6-tetrahydropyridine
4-hydroxyphenyllactate
4-O-Methyldopamine
-
0.069 mM, 50% inhibition
4-phenyl-1,2,3,6-tetrahydropyridine
5,5'-dithiobis(2-nitrobenzoate)
5-Hydroxydopamine
-
0.042 mM, 50% inhibition
6-Hydroxydopamine
-
0.029 mM, 50% inhibition
Adrenochrome
-
-
alpha-Methyltyrosine
-
1.1 mM, 50% inhibition
aminochrome
-
oxidation product of adrenaline, competitive vs. NADH
aminopterin
catecholamine
-
-
CdCl2
-
0.1 mM, complete inactivation
cis-Diaminodichloroplatinum
-
-
CoCl2
-
0.01 mM, 30% inhibition
dopachrome
-
0.6 mM, 50% inhibition
dopamine
epinephrine
-
0.13 mM, 50% inhibition
H2O2
regulatory function in vivo, above 0.03 mM, oxidation of Met146 and Met151 leads to inactivation of the enzyme due to disruption of the NADH-binding site
higenamine
-
noncompetitive vs. quinoid 2-amino-6,7-dimethyl-4-hydroxydihydropteridine and NADH
higenamine-1-carboxylic acid
-
noncompetitive vs. quinoid 2-amino-6,7-dimethyl-4-hydroxydihydropteridine
iodoacetamide
-
-
K2PtCl4
-
0.208 mM, half-life of inactivation: 3.1 min, NADH and 2 mM dithiothreitol completely protect
L-beta-3,4-dihydroxyphenylalanine
L-thyroxine
-
mixed-type inhibition
L-tyrosine
m-tyramine
methopterin
methotrexate
MgCl2
-
0.1 mM, complete inactivation
MnCl2
-
0.1 mM, complete inactivation
N-bromosuccinimide
-
-
N-ethylmaleimide
N-Methyldopamine
-
0.027 mM, 50% inhibition
NAD+
-
competitive vs. NADH
Noradrenalin
noradrenaline
-
-
norepinephrine
-
0.2 mM, 50% inhibition
o-hydroxyphenylacetic acid
octopamine
-
0.19 mM, 50% inhibition
p-chloromercuribenzoate
phenylacetic acid
phenylalanine
-
-
Phenyllactate
-
-
phenylpyruvate
quinonoid 2,4-diamino-7,8-dihydropteroylglutamic acid
-
-
quinonoid 6,6,8-trimethyl-7,8-dihydro(6H)pterin
-
0.2 mM, 50% inhibition
quiononoid 2,4-diamino-7,8-dihydropteroylglutamic acid
-
-
salsolinol
-
noncompetitive vs. quinoid 2-amino-6,7-dimethyl-4-hydroxydihydropteridine
serotonin
-
-
tetrahydrobiopterin
-
above 0.05 mM, substrate inhibition
trans-Diaminodichloroplatinum
-
-
trans-Pt(NH3)Cl2
-
1 mM, 96% inactivation after 2 h
tryptophan
-
-
tyramine
[4-hydroxyphenyl]acetic acid
-
-
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.036
2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydro-pteridine
0.34
2-Amino-4-hydroxy-6,7-dimethyl-quinonoid-dihydropteridine
-
-
0.0152
2-amino-4-hydroxy-6,7-dimethyldihydropteridine
-
cofactor NADH
-
0.017
5,6,7,8-tetrahydrobiopterin
-
37C, pH 7.2
0.0063 - 0.0152
6,7-dimethyl-5,6,7,8-tetrahydrobiopterin
0.012 - 0.015
6,7-dimethyl-5,6,7,8-tetrahydropterin
0.03
6,7-dimethyldihydropterin
-
-
0.035
6-Methyl-dihydropterin
-
-
0.366
6-methyl-quinonoid 7,8-(6H)-dihydropterin
-
-
0.0014 - 0.013
6-methyl-tetrahydropterin
0.03
6-methyldihydropterin
-
-
0.16
biopterin
-
-
0.754
cis-6,7-dimethyl-quinonoid 7,8-(6H)-dihydropterin
-
-
0.004
dihydrobiopterin
1.45
L-threo-neopterin
-
-
0.00079 - 2.9
NADH
0.0014 - 0.77
NADPH
0.018
quinonoid 2-amino-4-hydroxy-6,7-dimethyl-7,8-dihydropteridine
-
recombinant enzyme
0.006 - 0.34
quinonoid 2-amino-4-hydroxy-6,7-dimethyldihydropteridine
0.4
Quinonoid 6,6-dimethyldihydropterin
0.008 - 0.46
quinonoid 6,7-dimethyldihydropteridine
0.0008 - 0.0063
quinonoid 7,8-(6H)-dihydrobiopterin
0.669
quinonoid 7,8-(6H)-dihydropterin
-
-
0.0014 - 0.013
quinonoid 7,8-dihydro-6-methylpterin
0.00015 - 0.0016
quinonoid 7,8-dihydrobiopterin
0.0068
quinonoid 7,8-dihydropterin
-
cofactor NADPH, NADPH-specific enzyme
0.01
quinonoid dihydro-6,7-dimethyldihydropterin
-
enzyme from brain
-
0.028
quinonoid dihydrobiobiopterin
-
K150E mutant enzyme
0.00032 - 1
quinonoid dihydrobiopterin
1.32
Quinonoid dihydromonapterin
-
-
1.05
Quinonoid dihydroneopterin
-
-
1.45
quinonoid L-threo-dihydroneopterin
-
-
0.525
quinonoid-6,7-dimethyl-7,8-dihydropterin
-
-
0.128
quinonoid-6-methyl-7,8-dihydrobiopterin
-
-
0.0049 - 0.48
quinonoid-6-methyl-7,8-dihydropterin
0.0009 - 0.252
quinonoid-7,8-dihydrobiopterin
0.0075 - 0.144
quinonoid-7,8-dihydropterin
0.0034
quinonoid-dihydro-6-methylpterin
-
-
0.0009 - 0.017
tetrahydrobiopterin
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
510
2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine
Homo sapiens
-
-
17 - 280
NADH
20
NADPH
Homo sapiens
-
recombinant enzyme
162
quinoid 2-amino-4-hydroxy-6,7-dimethyl-7,8-dihydropteridine
Homo sapiens
-
recombinant enzyme
2 - 153
quinoid 6,7-dimethyldihydropteridine
0.3 - 227
quinoid 6-methyl-7,8-dihydropterin
16.7 - 52.2
quinoid 7,8-dihydrobiopterin
15.7 - 51.2
quinoid 7,8-dihydropterin
269
quinoid dihydrobiobiopterin
Rattus norvegicus
-
K150E mutant enzyme
1.7 - 200
quinoid dihydrobiopterin
10.9 - 113
quinonoid 7,8(6H)-dihydropterin
23.7 - 113
quinonoid 7,8-(6H)-dihydrobiopterin
20
quinonoid dihydrobiopterin
Pseudomonas sp.
-
-
200
tetrahydrobiopterin
Homo sapiens
-
-
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0019
(R)-2,10,11-trihydroxyapomorphine
0.0012 - 0.0022
(R)-apomorphine
0.0013 - 0.0022
(R)-N-n-propylaporphine
0.0028
1-methyl-4-(4'-hydroxyphenyl)-1,2,3,6-tetrahydropyridine
-
noncompetitive inhibition
0.023
2,4-diamino-7,8-dihydropteroylglutamic acid
-
-
0.0295
2,4-diaminopteroic acid
-
-
0.024
2,4-diaminopteroylglutamic acid
-
-
0.014 - 0.031
3',4'-deoxynorlaudanosoline-carboxylic acid
0.0037
3-(4-hydroxyphenyl)pyruvate
-
-
0.024
6-Hydroxydopamine
-
noncompetitive vs. quinonoid 2-amino-6,7-dimethyl-4-hydroxydihydropteridine and NADH
0.0049
Adrenochrome
-
-
0.01 - 0.21
aminopterin
0.006 - 0.014
dopamine
0.11 - 0.14
epinephrine
0.0015 - 0.0023
higenamine
0.0045
higenamine-1-carboxylic acid
-
vs. quinonoid 2-amino-6,7-dimethyl-4-hydroxydihydropteridine
0.26
L-Dopa
-
-
0.016
L-thyroxine
-
-
0.25
L-tyrosine
-
-
0.067
m-tyramine
-
-
0.025 - 0.26
methotrexate
0.0032
NADP+
0.49
noradrenaline
-
noncompetitive inhibition
0.016 - 0.19
norepinephrine
1
phenylalanine
0.05
phenylpyruvate
-
mixed inhibition
0.01
phenylpyruvic acid
-
uncompetitive vs. quinonoid 2-amino-6,7-dimethyl-4-hydroxydihydropteridine
0.022
quinonoid 2,4-diamino-7,8-dihydropteroylglutamic acid
-
-
0.09
salsolinol
-
-
1.2
serotonin
-
competitive inhibition
0.2
tryptophan
-
mixed inhibition
0.024 - 0.05
tyramine
0.074
[4-hydroxyphenyl]acetic acid
-
-
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.04
tyramine
Homo sapiens
-
IC50: 0.04 mM
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.069
enzyme extract of microplasmodia
0.431
-
NADPH-specific enzyme from liver
0.706
-
NADPH-specific liver enzyme
2.51
-
-
5.2
-
recombinant enzyme, substrate dihydrofolate
20.2
-
NADH-specific liver enzyme
24
-
NADH-specific liver enzyme
38.3
purified recombinant enzyme
56
-
T119 insert mutant enzyme
125.8
-
recombinant enzyme
224
-
enzyme from brain
300
-
W104G mutant enzyme
412
-
recombinant enzyme
610
-
enzyme from brain
800
-
recombinant enzyme
1180
-
enzyme from brain
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.8
-
assay at
7.5
-
assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.8 - 7.8
-
pH 6.8: about 45% activity, pH 7.8: about 15% activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.6 - 4.7
-
isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
cell culture
Manually annotated by BRENDA team
-
low activity
Manually annotated by BRENDA team
-
continuous
Manually annotated by BRENDA team
-
low activity
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
24600
-
calculated, monomer
25000
-
SDS-PAGE, monomer
41000
-
gradient PAGE
43500 - 49000
-
gel filtration
46000
about, recombinant enzyme, gel filtration
47000
-
gel filtration
49000 - 50000
-
NADH-dihydropteridine reductase
51000
-
gel filtration
55000
-
gel filtration
65000
-
equilibrium centrifugation, NADPH-specific liver enzyme
68000
-
NADPH-specific enzyme, gel filtration
69000
-
NADPH-specific liver enzyme, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
-
x * 24528, deduced from nuclotide sequence
oligomer
-
x * 23000, recombinant enzyme, SDS-PAGE
additional information
-
x * 25000, SDS-PAGE, weak band at 50000 Da which possibly corresponds to the dimeric form of the enzyme, calculated mass of the His-tagged protein is 24200 Da
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
silky crystals in the presence or absence of NADH upon a slow increase of ammonium sulfate concentration up to 50-55% at pH 6.5
-
complexed with NAD+, hanging drop vapor diffusion method, using 17% (w/v) polyethylene glycol 3350 and 100 mM Bis-Tris at pH 6.5
dicDHPR-NAD complex by incubation in 20 mM Tris-HCl, pH 8.0, hanging-drop vapor diffusion (4 microl drops), 18C (291 K), screening solution (0.1 M bis-tris, pH 6.5, 17% PEG 3350), hexahistidine tag not removed, x-ray diffraction
-
hanging drop vapour diffusion, polyethylene glycol 4500, various concentrations of Tris phosphate buffer, pH 7.8, 8% methanol and 1 mM beta-mercaptoethanol, 2.5 A resolution
-
hanging drop vapour diffusion, 2 crystal forms depending on whether inorganic salts or alcohols are used as precipitating agents, crystals diffract to at least 2.3 A
-
hanging drop vapour diffusion, polyethylene glycol 4500, Tris phosphate, pH 7.8, 10% methanol, 4C, crystals of binary complexes with NADH of recombinant wild-type, W86I, Y146F, Y146H, Y146F/K150Q and A133S mutant enzymes, 2.1-2.3 A resolution
-
selenomethionine-substitued enzyme in complex with inhibitor methotrexate or substrate dihydrofolate
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 8
-
unstable below and above
484992
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40
-
5 min, 80% loss of activity, EDTA protects
80
-
1 min, inactivation
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
NADH protects against inactivation
-
NADPH protects against inactivation
-
not stable during gel filtration
-
stable to freezing at -70C and thawing
-
unstable to (NH4)2SO4 precipitation
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, stable for at least a year, enzyme activity in ammonium sulfate precipitate
-
-70C, stable for at least 2 years
-
-80C, 40 mM potassium phosphate buffer, pH 6.8, stable for at least a week
-
-80C, stable for at least 3 months
-
-80C, stable for years
-
4C, stable for up to 2 weeks
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ATCC 11299a
-
cells centrifuged, washed with PBS (pH 7.4), resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5), disrupted by sonication, centrifugation, supernatant filtered (0.45 microm), applied to a column with nickel-NTA beads, eluted (50 mM Tris-HCl, pH 7.5), active fractions pooled, concentrated, and exchanged into 50 mM sodium phosphate, pH 6.8, ultrafiltration, applied to a cation-exchange chromatography HS20 column, eluted with salt gradient at pH 6.8, concentration by ultrafiltration, gel-filtration chromatography with Superdex 200 column in 50 mM Tris-HCl, pH 8.0, concentration of eluted fractions in 20 mM Tris-HCl, pH 8.0, by ultrafiltration
-
homogenization of smooth muscle strips in 2-DE lysis buffer containing 8 M urea, 2 M thiourea, 65 mM DTT, 2% CHAPS, and protease inhibitor cocktail, centrifugation, supernatant used for 2-DE and MALDI-TOF/TOF-MS
-
naphthoquinone affinity chromatography, 5'-AMP-Sepharose
-
native enzyme and recombinant wild-type, W104F, A6V and D37I mutant enzyme
-
nickel-affinity column chromatography
recombinant enzyme
-
recombinant enzyme from Escherichia coli by ion exchange chromatography and gel filtration to homogeneity
recombinant enzyme, partially from Escherichia coli, solubilization at pH 10.0
-
recombinant protein using His-tag
-
recombinant wild-type and several mutant enzymes
-
recombinant wild-type, G19D, W104G and T119insert mutant enzymes
-
simple two step procedure i.e. affinity chromatography on Matrex gel blue A and hydrophobic chromatography on phenyl-Sepharose
wild-type and recombinant Y150H, Y150S, Y150F, Y150E, Y150K, G151S and G23D mutant enzymes
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning of full-length cDNA, expression in COS cells
-
dicDHPR gene (residues 1-231) cloned into pET-28b expression plasmid with hexahistidine tag and thrombin cleavage site, expression in Escherichia coli BL21 (DE3)
-
DNA sequence determination and analysis, overexpression in Escherichia coli
-
expressed as His-tag fusion protein in Escherichia coli
-
expressed in HEK-293T cells
-
expression of cloned cDNA in Escherichia coli
-
expression of wild-type and several mutants in Escherichia coli
-
expression of wild-type, G19D, W104G and T119insert mutant enzymes in Escherichia coli
-
expression of wild-type, Y150H, Y150S, Y150F, Y150E, Y150K, G151S and G23D in Escherichia coli
-
high-level expression in Escherichia coli
-
liver enzyme expressed in Escherichia coli
-
recombinant expression in Escherichia coli
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
enzyme expression is significantly decreased in the renal cortex of diabetic rats
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
G151S
-
mutation found in children suffering from phenylketonuria due to enzyme deficiency
G19D
-
completely inactive
G23D
-
mutation found in children suffering from phenylketonuria due to enzyme deficiency, inactive mutant enzyme
T119insert
-
reduced activity
W104G
-
similar activity as wild-type
Y150E
-
lower kcat with 6-methyl-7,8-dyhydropterin than wild-type
Y150F
-
lower kcat with 6-methyl-7,8-dyhydropterin than wild-type
Y150H
-
lower kcat with 6-methyl-7,8-dyhydropterin than wild-type
Y150K
-
lower kcat with 6-methyl-7,8-dyhydropterin than wild-type
Y150S
-
lower kcat with 6-methyl-7,8-dyhydropterin than wild-type
A6V
-
no significant differences to wild-type
K150I
-
lower kcat for quinonoid dihydrobiopterin than wild-type
K150M
-
lower kcat for quinonoid dihydrobiopterin than wild-type
K150Q
-
lower kcat for quinonoid dihydrobiopterin than wild-type
N186A
-
lower kcat for quinonoid dihydrobiopterin than wild-type
W104F
-
no significant differences to wild-type
W86I
-
lower kcat for quinonoid dihydrobiopterin than wild-type
Y146F/A133S
-
almost no activity with quinonoid 6,7-dimethyldihydropteridine, higher activity with quinoid dihydrobipterin than wild-type
Y146F/K150E
-
no activity with quinonoid 6,7-dimethyldihydropteridine, higher activity with quinoid dihydrobipterin than wild-type
Y146F/K150Q
-
lower kcat for quinonoid dihydrobiopterin than wild-type
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
-
enzyme deficiency leads to progressive mental and physical retardation despite dietary phenylalanine restriction. Overview on genomic structure and location of natural mutations in the QDPR gene coding for enzyme
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